Blood platelets play an essential role in hemostasis, as well as in the pathophysiology of thrombosis. The purpose of this proposal is to investigate the cytoskeletal mechanics of platelet formation. Although it is well established that platelets originate from megakaryocytes, many unanswered questions remain regarding the mechanics by which platelets are formed and released. The favored model of platelet formation recognizes that terminally differentiated megakaryocytes extend long cytoplasmic processes, designated proplatelets, which function as essential intermediate structures in platelet biogenesis. We have recently discovered a new intermediate stage in platelet production, the preplatelet, and have established that microtubules and the spectrin-based membrane skeleton are involved in platelet production. How preplatelets convert into platelets, however, is not known, and many of the molecular details underlying the contribution of the cytoskeleton to platelet production remain to be uncovered. The three Specific Aims of this proposal focus on the role of the cytoskeleton in platelet production. Specific Aim 1 will examine how microtubules convert preplatelets into barbell shapes that divide to release platelets, with the goal of testing the hypothesis that microtubule sliding powers preplatelet fission and release. The role of specific microtubule-associated proteins will be established. Specific Aim 2 will define how the spectrin-based membrane skeleton contributes to proplatelet production. Using a newly developed permeabilized proplatelet system, the spatial and temporal localization, as well as the function of key membrane skeleton proteins will be established. The role of the Filamin-GPIba-actin linkage in the preplatelet to platelet transition will be tested. Finally, Specific Aim 3 will evaluate the importance of microtubules in forming the demarcation membrane system, with the goal of using an in vitro model to determine if the demarcation membrane system forms by membrane- associated motors moving over microtubules or by the attachment to the tips of elongating microtubules. These experiments will define the function of specific cytoskeletal proteins in demarcation membrane system formation. Taken together, we expect that findings made as a result of this investigation will provide an improved understanding of the molecular mechanisms that regulate platelet formation, and lay the foundation for novel therapeutic approaches to accelerate platelet production in patients with thrombocytopenia, as well as strategies aimed at improving the in vitro generation of platelets for infusion. PUBLIC HEALTH RELEVANCE: Blood platelets play a critical role in stimulating clot formation and repair of vascular injury. Life-threatening thrombocytopenia (low platelet count) can occur in patients for a variety of reasons, including chemotherapy, radiation treatment, trauma, severe burns, organ transplant surgery, and genetic disorders. The studies proposed in this application will advance our understanding of blood platelet production and may lead to improved therapies for accelerating platelet production in these patients and lay the groundwork for the development of in vitro production of platelets for infusion.